R.J. van Dorssen

Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II

The D1-D2-cytochrome b-559 reaction center complex and the 47 kDa antenna chlorophyll protein isolated from spinach Photosystem II were characterized by means of low temperature absorption and fluorescence spectroscopy. The low temperature absorption spectrum of the D1-D2-cytochrome b-559 complex showed two bands in the Qy region located at 670 and 680 nm. On the basis of its absorption maximum and orientation the latter component may be attributed at least in part to P-680, the primary electron donor of Photosystem II. The 47 kDa antenna complex showed absorption bands at 660, 668 and 677 nm and a minor component at 690 nm. The latter transition appeared to be associated with the characteristic low temperature 695 nm fluorescence band of Photosystem II. The 695 nm emission band was absent in the D1-D2 complex, which indicates that it does not originate from the reaction center pheophytin, as earlier proposed. The transition dipole responsible for the main fluorescence at 684 nm from this complex had a parallel orientation with respect to the membrane plane in the native structure. The reaction center preparation contains two spectrally distinct carotenoids with different orientations.

Spectroscopic properties of chloroplast grana membranes and of the core of photosystem ii

An oxygen-evolving Photosystem II core complex essentially free of the light-harvesting chlorophyll ab protein complex, containing 45 chlorophylls per reaction center was isolated from spinach chloroplasts. Its structural integrity was established by studying its photochemistry and spectral properties. The absorption spectrum measured at 4 K revealed the presence of at least five spectrally distinct chlorophyll a species. The same bands, but in different proportions, were observed in a Photosystem II grana preparation used as starting material for the preparation of the core complex. The relative contributions of these components to the overall absorption were calculated by deconvoluting this spectrum into Gaussian bands. The core complex was enriched in a long-wave band located at 683 nm, which presumably reflects the presence of 8–10 pigment molecules that are closely associated with the reaction center. Low temperature fluorescence emission spectra showed the characteristic Photosystem II emission bands located at 685 nm (F685) and at 695 nm (F685). The two states giving rise to these emissions are in thermal equilibrium down to 70 K. It is suggested that F685 arises from a chlorophyll a species absorbing at 676 nm and that F695 is the result of fluorescence from the photoactive pheophytin a absorbing around 683 nm.

Optical and structural properties of chlorosomes of the photosynthetic green sulfur bacterium Chlorobium limicola

Abstract Isolated chlorosomes of the photosynthetic green sulfur bacterium Chorobium limicola upon cooling to 4 K showed, in addition to the near-infrared absorption band at 753 nm due to bacteriochlorophyll c , a weak band near 800 nm that could be attributed to bacteriochlorophyll a . The emission spectrum showed bands of bacteriochlorophyll c and a at 788 and 828 nm, respectively. The fluorescence excitation spectrum indicated a high efficiency of energy transfer from bacteriochlorophyll c to bacteriochlorophyll a . When all bacteriochlorophyll c absorption had been lost upon storage, no appreciable change in the optical properties of the bacteriochlorophyll a contained in these ‘depleted chlorosomes’ was observed. The fluorescence and absorption spectra of the chlorosomal bacteriochlorophyll a were clearly different from those of the soluble bacteriochlorophyll a protein present in these bacteria. The results provide strong evidence that bacteriochlorophyll a , although present in a small amount, is an integral constituent of the chlorosome. It presumably functions in the transfer of energy from the chlorosome to the photosynthetic membrane; its spectral properties and the orientation of its near-infrared optical transitions as determined by linear dichroism are such as to favor this energy transfer.

Spectroscopic properties of antenna complexes of Rhodobacter sphaeroides in vivo

Abstract Intact membranes of antenna mutants of Rhodobacter sphaeroides obtained by chemical mutagenesis containing only the B800–850 or the B875 reaction center complex were used to study the spectral properties of these antenna complexes separately in vivo. Wild-type spectral characteristics were restored to each mutant, following complementation by the relevant gene. It is shown that the absorption spectra of recombinant strains and of wild-type Rhodobacter sphaeroides can be analyzed in terms of those of the separate complexes as observed in the mutants. Distinct differences occur between the spectra of the antenna complexes isolated by means of detergent solubilization of the membrane and those of the mutants. Measurements of absorption and flash-induced absorption difference spectra and of linear dichroism and fluorescence polarization spectra at low temperature indicate that in the intact membrane the previously characterized bacteriochlorophyll Q y absorption bands near 800, 850 and 875 nm display an optical inhomogeneity and that they all contain a relatively weak transition at longer wavelength, the orientation of which is more parallel to the membrane plane than the orientation of the main transitions. Rapid and efficient energy transfer to the long-wave component (BChl 870 ) in the B800–850 complex could be demonstrated. Some of the long-wave transitions are also observable at room temperature. They may reflect the mode of aggregation of the complexes in their lipid environment and, by increased overlap between donor emission and acceptor absorbance, serve to facilitate energy transfer within the antenna system.

Excitation trapping and charge separation in Photosystem II in the presence of an electrical field

Abstract To investigate the effects of a membrane potential on excitation trapping and charge separation in Photosystem II we have studied the chlorophyll fluorescence yield in osmotically swollen chloroplasts subjected to electrical field pulses. Significant effects were observed only in those membrane regions where a large membrane potential opposing the photochemical charge separation was built up. When the fluorescence yield was low, close to F 0 , a much higher yield, up to F max , was observed during the presence of the membrane potential. This is explained by an inhibition by the electrical field of electron transfer to the quinone acceptor Q, resulting in a decreased trapping of excitations. A field pulse applied when the fluorescence yield was high, Q and the donor side being in the reduced state, had the opposite effect: the fluorescence was quenched nearly to F 0 . This field-induced fluorescence quenching is ascribed to reversed electron transfer from Q − to the intermediate acceptor, pheophytin. Its field strength dependence suggests that the midpoint potential difference between pheophytin and Q is at most about 300 mV. Even then it must be assumed that electron transfer between pheophytin and Q spans 90% of the potential difference across the membrane.

Antenna organization and energy transfer in membranes of Heliobacterium chlorum

Abstract Absorption, fluorescence emission and fluorescence excitation spectra of membranes of the recently discovered photosynthetic bacterium Heliobacterium chlorum (Gest, H. and Favinger, J.L. (1983) Arch. Microbiol. 136, 11–16) showed that at 4 K at least three spectroscopically different forms of bacteriochlorophyll g (BChl g 778, BChl g 793 and BChl g 808) can be discerned in the antenna system. Efficient energy transfer occurs from the short-wave-absorbing bacteriochlorophylls to BChl g 808. Energy transfer to bacteriochlorophyll, albeit with lower efficiency (70%), also occurred from the main carotenoid, neurosporene, and from a pigment absorbing at 670 nm. The complex structure of the antenna system is also reflected by fluorescence polarization and linear and circular dichroism spectra. Significant circular dichroism was only observed for BChl g 793, and different orientations were observed for the various Q y transition dipoles, the one of BChl g 808 making a smaller angle with the plane of the membrane than those of the other bacteriochlorophylls.

The construction and properties of a mutant of rhodobacter sphaeroides with the lh1 antenna as the sole pigment protein

Recent work on the spectroscopic properties of the B875 (LH1) antenna of Rb. sphaeroides has been performed on the complex purified from membranes of the wild-type solubilised in lithium dodecyl sulphate. In order to facilitate an examination of the properties of the membrane-bound antenna free from other complexes, mutant M2192 was constructed from mutant M21, which contains reaction centres and LH1 but lacks the B800–850 (LH2)complex. This was accomplished by insertion of transposon Tn5 into the puf L gene which encodes the L polypeptide of the reaction centre. This manipulation leaves B875 as the sole pigment protein, which has been confirmed by Southern and Northern hybridisation, gel electrophoresis and fluorescence and absorbance spectroscopy. Evidence from Gaussian deconvolution of the Qy absorbance region, and from fluorescence polarisation, suggests that the long-wavelength species BChl 896 is present, and may be an inherent property of the LH1 antenna.

Charge separation and trapping efficiency in membranes of Heliobacterium chlorum at low temperature

Photooxidation of the primary electron donor, P-798, and the transfer of excitation energy to the reaction center in membranes of Heliobacterium chlorum were studied at low temperature. The difference spectrum of P-798 photooxidation at 5 K is dominated by a bleaching at 793 nm. P-798 + was rereduced after a flash with a time constant of 2.3 ms, presumably by a back reaction with a reduced electron acceptor. Analysis of the kinetics at various wavelengths showed the contribution of two othercomponents to the absorbance changes at 5 K. The first one could be attributed to the photooxidation, within 10 μs, of bound cytochrome c -553 in about 3% of the reaction centers; the second one to the generation of the triplet states of P-798 and bacteriochlorophyll (BChl) g with (average) lifetimes of 350 ± 50 μs.Measurements of the relative efficiency of energy transfer from the antenna to the reaction center indicated that this transfer is still quite efficient at 5 K and essentially equal for the three BChl g species, BChl g 778, BChl g 793 and BChl g 808. The function of the latter pigment thus may be to focus the excitation energy on pigments closeto the reaction center, similar to what has been proposed for long-wave components in the antenna of purple bacteria. However, in H. chlorum this process would appear to imply ‘uphill’ transfer to P-798. Possible explanations to avoid this difficulty are discussed.

Non-electrogenic charge recombination in Photosystem II as a source of sub-millisecond luminescence

The luminescence emitted by Photosystem II at times between 1 μs and 1 ms after flash illumination was studied. Membrane potentials of up to 1 V, generated in osmotically swollen chloroplasts by externally applied electrical field pulses, had no effect on the decay components of this luminescence. Instead the field induced an additional, much slower luminescence due to a stimulated recombination of the primary charge separation in Photosystem II centers. The field-insensitive luminescence, in contrast to the field-induced luminescence, was independent of the redox state of the primary acceptor Q, and appeared not to originate from the same Photosystem II centers. The decay kinetics consisted mainly of two phases with 10 and 60 μs halftimes. The initial amplitude could be restored at all times by a second flash, indicating that both phases are due to a reversal of the field-insensitive charge separation. In the presence of DCMU and hydroxylamine, no luminescence was observed between 10 μs and 1 ms after the flash. Presumably the reoxidation of the electron acceptor was significantly slowed down, but it was still much faster than that of Q−.

The Effects of an Electrical Field on the Primary Reactions of System II

When osmotically swollen chloroplasts (blebs) are subjected to an externally applied electrical field pulse, a greatly enhanced local field is generated in the membrane. In this way a fraction of system II can be exposed to membrane potentials of up to 1 V with sub-ms time resolution. It was reported before that the charge recombination after illumination of system II in the presence of DCMU could be accelerated by many orders of magnitude (De Grooth, Van Gorkom, 1981). We now report field-induced changes of the chlorophyll fluorescence yield which suggest that the field-sensitive reaction is the electron transfer between the ‘primary’ acceptor Q and the ‘intermediary’ acceptor Pheophytin (at least in those system II centers that are membrane potential-sensitive at all, see accompanying paper).